US4859594A - Microorganisms for biodegrading toxic chemicals - Google Patents
Microorganisms for biodegrading toxic chemicals Download PDFInfo
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- US4859594A US4859594A US06/918,840 US91884086A US4859594A US 4859594 A US4859594 A US 4859594A US 91884086 A US91884086 A US 91884086A US 4859594 A US4859594 A US 4859594A
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/02—Aerobic processes
- C02F3/10—Packings; Fillings; Grids
- C02F3/102—Permeable membranes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/858—Methylomonas
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/874—Pseudomonas
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/874—Pseudomonas
- Y10S435/876—Pseudomonas fluorescens
Definitions
- This invention relates to novel microorganisms, to a process for immobilizing and affixing these microorganisms to substrates for use as biocatalysts, to the novel biocatalyst compositions so produced, and to the use of the novel biocatalyst compositions for conducting chemical reactions, especially for the detoxification of toxin-polluted streams.
- cross-linking Methods have been described in the literature for the immobilization of enzymes, microbial cells, plant cells and animal cells. In general, such methods have been classified as cross-linking, entraping and carrier-binding.
- the cross-linking method is based on the formation of chemical bonds, the immobilization of the enzymes or cells being accomplished by the formation of intermolecular cross-linkages between the enzyme molecules or the cells, by means of bifunctional or multi-functional reagents.
- Cross-linking reagents have included such compounds as glutaraldeyde, bisisoocyanate derivative and bisdiazobenzidine.
- the entrapping method is based on a technique of confining the enzymes or cells in the lattice of a polymer matrix, or enclosing them in semipermeable membranes.
- the enzyme or cell itself is not physically bound to the gel matrix or membrane.
- the enzymes or cells are linked directly to water-insoluble carriers, e.g., polysaccharides (cellulose, dextron, and agarose derivatives), proteins (gelatin and albumin), synthetic polymers (ion-exchange resin and polyvinylchloride), and inorganic materials (brick, sand and glass).
- water-insoluble carriers e.g., polysaccharides (cellulose, dextron, and agarose derivatives), proteins (gelatin and albumin), synthetic polymers (ion-exchange resin and polyvinylchloride), and inorganic materials (brick, sand and glass).
- water-insoluble carriers e.g., polysaccharides (cellulose, dextron, and agarose derivatives), proteins (gelatin and albumin), synthetic polymers (ion-exchange resin and polyvinylchloride), and inorganic materials (brick, sand and glass).
- the primary objective of the present invention to overcome these and other disadvantages by providing novel microorganisms, process for the immobilization and attachment of these and other microorganisms to a carrier, or support, to form a biocatalyst, and process for the passage of a chemical-containing effluent stream through a bed of said catalyst to conduct biochemical reactions.
- a further, and more specific object is to provide novel microorganisms on immobilizing solid supports suitable for conducting continuous biochemical reactions, especially low energy biochemical reactions useful for the detoxification of toxic chemical-containing effluents brought into contact therewith at high detoxification rates.
- a more specific object is to provide novel microorganisms, and biocatalyst compositions produced from such organisms which can effectively biotransform and biodegrade a wide class of toxicants within an acceptable time frame.
- microorganisms isolated from natural environments, purified and genetically modified, to provide novel microbial strains.
- novel microbial strains or the enzyme fractions thereof can be immobilized by bonding same to porous water-insoluble solid adsorbents to form biocatalytic compositions which, when used as biocatalyst beds, can be contacted with toxic effluent streams for the detoxification thereof.
- novel microorganisms of this invention are characterized as (1) Pseudomonas fluorescens Ppr S-4-1 (ATCC SD 904); (2) Pseudomonas fluorescens Chemostat 5 1/2 mo (ATCC SD 903); (3) Pseudomonas cepacia Sys 2-A (ATCC SD 905); (4) Methylobacter rhodinum SC 971-Red (ATCC 113-X); and (5) Methylobacter species SC 971-White (ATCC 138-X) [Bergy's Manual of Systematic Bactoriology, Vol. 1, Krieg and Holt, Williams & Williams Baltimore, Md., U.S.A. and London, England (1984).]
- the Pseudomonas fluorescens Pr S-4-1 was obtained from a field sample of a sediment taken from Capital Lake, a fresh water lake in Baton Rouge, Louisiana, at a temperature of 23° C. The sediment was heavily contaminated with PCB's.
- the Pseudomonas fluorescens Chemostat 5 1/2 mo was isolated from a field sample of sediment obtained from an abandoned waste site in Ascension Parish, near Darrow, Louisiana, heavily contaminated by chlorinated ethanes.
- the Pseudomonas cepacia Sys 2-A was isolated from a field sample of a sediment obtained at Shell Beach, Louisiana, a Mississippi River Gulf outlet.
- the sediment was heavily contaminated with polychlorophenols (PCP's), had a salinity of 12 ppm, and was obtained at 18° C.
- PCP's polychlorophenols
- the Methylobacter cultures i.e., Methylobacter rhodinum SC 971-Red (ATCC 113-X) and Methylobacter species SC 971-White (ATCC 138-X)
- the activated charcoal was heavily contaminated with Triazines, the Triazine herbicide being one of the herbicides most commonly used in American agriculture. Triazines are of a recalcitrant nature and are not successfully degraded by conventional waste treatment processes.
- a field sample of each of the five specimens was treated in the laboratory, continuous flow microcosms of each being formed and treated as follows:
- PCBs polychlorinated biphenyls
- 5 ppm same as PCP
- final conc 50 ppm of either Aroclor 1232, 1242, 1248, 1254, 1260, or specific congenors/isomers of each.
- Total time 3 mos. Cultures maintained on 200 ppm PCB agar.
- microorganisms viz., (1), (2), (3), (4) and (5) respectively, were affixed to surfaces characterized as chitin, chitosan, cellulose, diatomaceous earth, porous inorganic oxides, e.g., alumina, and the like.
- a culture of each microorganism has been deposited in the American Type Culture Collection at Rockville, Maryland and each culture assigned an identifying number, each as previously identified, this depository affording permanence of the deposit and ready accessibility thereto by the public on grant of a patent, and under conditions which assure (a) that access to the culture will be available during pendency of the patent application to one determined to entitled thereto under 37 CFR 1.14 and 35 USC 122, and (b) that all restrictions on the availability to the public of the culture so deposited will be irrevocably removed upon grant of a patent.
- Progenitors of the microorganisms of this invention or others are selected from a soil, sediment, or stream enriched by accidental, purposeful or deliberate treatment with the toxicant for which the cultured genetically modified immobilized microorganism of this invention is to be used as a biocatalytic detoxicant.
- a progenitor microoganism which is to be cultured, genetically modified and immobilized upon a substrate for use in detoxifying streams which contain pentachlorophenol (PCP), is selected from a soil, sediment, or stream which has been heavily contaminated with PCP's, by accidental previous spillage(s) or deliberate treatments with PCP.
- the progenitor microorganism is isolated from a soil, sediment, or stream heavily contaminated with PCB's.
- the contaminated soil, sediment, or stream from the field environment, containing the progenitor microorganism is natural in all respects except arguably for the presence of the toxicant, or contaminant. It is placed in a microcosm which simulates, or duplicates to the fullest extent possible, except for the level of concentration of the toxicant itself, the conditions of the environment from which it was taken, viz., as regards pH, temperature, water composition, salinity, etc.
- the level of the toxicant maintained within the simulated environment, while initially not excessive of that of the sediment from which the specimen was taken, is gradually or incrementally increased. Observations are periodically, or continuously made of the microcosm to detect the different strains of microorganisms which survive these treatments, and the most hardy of these cultures are selected and grown on an agar surface which contains the same toxicant as the sole carbon source. Simultaneously, the agar surface is treated with a mutagen, or mutagens, to kill and destroy the least hardy of the microorganisms. Some survive; most do not.
- the now essentially pure, or sterile, cultures of a microorganism of this invention are now again placed in a microcosm and subjected to the same stress variables, or conditions found in the natural, or field environment from which they, in admixture with other microorganisms, were originally taken with the exception that the sterile cultures are again subjected to treatment with progressively increasing concentrations of the selected toxicant.
- the toxicant is substituted for the more conventional carbon source, and may be made eventually the sole carbon source for the microorganism.
- the concentration of the toxicant is increased to a level at least approximately equal to, and greater than that of the toxicant in the toxin-containing stream which is to be treated by the immobilized microorganism.
- the pH, temperature, water composition, salinity and the like, are maintained at essentially the same levels as in the environment from which the field sample was obtained.
- Nitrogen phosphorus and minerals from a suitable source as known desirable for the growth of healthy microorganisms, are added to the microcosm.
- the microorganisms of this invention are faculative anaerobes. They do not require oxygen, and hence oxygen need not be added to the microcosm to sustain healthy reproduction and growth.
- the pure microorganism cultures of this invention are in effect treated within the microcosm in an environment which simulates long term reproduction, and growth of the colonies.
- the microorganism adapts to the toxicant to which it is exposed and assimulates it as a preferential source of carbon.
- This type of environmental adaptation simulates generally many years of reproduction, adaptation and growth by the microorganism.
- the progenitor microorganism is genetically modified by such adaptation, and it becomes increasingly resistant to destruction and harm from the toxicant to which it is in constant exposure, and indeed becomes able to thrive on this new carbon source. Cultures of the genetically modified microorganism are subsequently separated from the microcosm, suitably by filtration.
- the microorganism, or another similarly obtained from a soil, sediment or stream, is then immobilized by attachment to a suitable substrate.
- a microorganism of this invention is immobilized upon a solid surface, or substrate to form a catalytic, or biocatalytic composition. This is accomplished generally by either of two procedures: (1) a substrate is first treated, impregnated, or coated with a solution which constitutes a carbon source, or additional carbon source where some carbon may be supplied by the substrate, and the microorganism is then brought in contact with the pretreated surface, or surface containing the added carbon source, or (2) the substrate is treated simultaneously with a solution of the carbon source and the microorganism, as by treatment of the substrate with an admixture of the carbon source and the microorganism.
- ingredients other than carbon needed for the growth of healthy microorganisms are added to the substrate by contact thereof with a solution containing the required nutrients, e.g., source of nitrogen, phosphorus and selected minerals as salts.
- a solution containing the required nutrients e.g., source of nitrogen, phosphorus and selected minerals as salts.
- the toxicant for which the microorganism has been grown for use as a detoxicant and on which the microorganism has learned to thrive as a co-carbon source is added to the treated substrate.
- the concentration of the toxicant co-carbon source can be gradually or incrementally increased, and finally completely substituted for the original carbon source applied during the immobilization procedure.
- biocatalyst thus formed by attachment to a substrate of a purified, genetically modified microorganism of this invention, or another similarly obtained from a soil, sediment or stream, having been deprived from normal carbon sources, and forced to accept and in fact thrive on a specific toxin as a substitute carbon-source, can now be employed as a particulate biocatalytic mass for use in the detoxification of streams polluted with similar toxins.
- a biocatalyst of this invention wherein the progenitor microorganism was obtained from a natural sediment contaminated with, e.g., PCB's, the progenitor microorganism cultivated in the presence of a mutagenic agent an increasing concentration of PCB's as a substitute carbon source to produce a purified, genetically modified microorganism of this invention, and this microorganism then affixed to a substrate to form said biocatalyst, can be used for the detoxification of PCB-containing streams.
- the new mutant microorganisms are thus developed from progenitor microorganisms which are deprived of the normal sources of carbon, and forced to take the carbon needed for their nutrition and energy from toxins.
- a mutant microorganism of this invention thus bred on a certain type of toxin, "learns" to accept and thrive on this toxin as its carbon source and, after having been affixed to a support, can effectively biotransform and biodegrade within an acceptable time period the toxin of a stream containing this toxin when the stream is contacted therewith.
- a biocatalyst formed from a purified, genetically modified microorganism of this invention, or another similarly obtained, can, e.g., be charged as a particulate mass into a tubular vessel and retained or packed therein as a bed.
- a microorganism separated, cultivated and obtained from a sediment contaminated with, e.g., pentachlorophenol, PCP can be affixed upon a substrate to form the biocatalytic composition, and the biocatalyst used for the detoxification of a PCP-containing effluent.
- Certain progenitor Pseudomonas and Methylobacter microorganism species of this invention were isolated from toxic-contaminated sediments from various Louisiana locations, as identified in Table 3 below, and colonized in an agar medium by subjecting an initially mixed microbial population from said source to ever increasing concentrations of a toxicant similar to that found in said contaminated sediment, in the presence of U.V. radiation as a mutating agent. Each of the microorganism strains were then repeatedly isolated after treatments at increasingly elevated toxicant levels, and exposure to U.V. radiation, during which the microorganisms species of this invention thrived, and grew as other microorganisms were killed.
- Cultures of the microorganisms of this invention were then further colonized in a minimal salt solution broth containing the toxin.
- the minimal salt solution was sterilized by autoclaving, the toxin was filter sterilized and aseptically added to the sterile broth, and the microbial strain was grown on a rotary platform shaker in the minimal salt solution broth containing 0.1% of the toxin.
- Residual and insoluble toxin components were removed by filtration, and the cells were resuspended in a 0.025 MPO 4 buffer (pH 7.0) solution. Cells were harvested as biologically pure cultures from the late exponential growth phase by centrifugation at 10,000 xg for 10 minutes.
- the cultivated biologically pure microorganism was then attached to a substrate, and thereby immobilized to form a biocatalytic composition.
- Various substrates were employed some of which inherently contained a source of carbon, and others to which a carbon source was added prior to or at the time the microorganism was affixed thereto.
- a preferred substrate is chitin or chitosan, an aminopolysaccharide.
- chitosan was employed as a component of a substrate, chitosan having been derived from the fresh water crayfish Procambarus clarkii by removal of the chitinous exoskeleton. The chitinous exoskeleton was dried at 60° C.
- Celite particles of chitosan was first pretreated with solution of the microbe in said previously described minimal salt solution containing the required growth factors, and 0.1% of the toxin, the microorganism attaching to the Celite-chitosan substrate to form a microbial mat comprising a biologically pure culture of the cultivated microorganism.
- the bed of chitinous substrate to which the microorganism attached was flushed from time to time to remove non-attached microorganisms, and the concentration of the toxin in the minimal salt solution broth with which the cultivated microorganism was maintained in contact was gradually increased from a 0.1% level over a number of sequential treatments to a 100% level of the toxin.
- substrates were employed which did not contain a natural carbon source
- carbon was added by pretreatment of the substrate with a 1% solution of gluteraldehyde.
- the microorganism was then added to the substrate to form the microbial mat. Thereafter the substrate was flushed to remove non-attached microorganisms for maximizing active sites, and the substrate treated with solutions of the required nutrients, inclusive of the toxin, the toxin having been added in incrementally increasing concentrations to provide a new carbon source to which the microorganism adapted, and thrived thereupon.
- the substrate with its glycocalyx microbial mat thus formed thereupon, and adapted to accept the toxin as a satisfactory source of carbon, was thus formed into a biocatalytic composition ready for use in the removal from toxin-contaminated streams of the toxins, on which it has learned to thrive.
- the biocatalytic composition thus formed was then charged to a tubular reactor provided with an inlet and outlet.
- the biocatalytic composition was contained therein as a packed bed. Influents with varying types of toxins, in differing concentrations were then introduced into the reactor at differing void volumes and flow rates, with the results described in Table 3.
- the first column identifies each of the microorganisms employed in the tests
- the second column identifies the environmental source from which the miccroorganism was obtained
- the third column the isolation enrichment media employed to produce the biologically pure culture of the respective microorganism from which a biocatalytic composition was made by affixing the respective organism to a support.
- the fourth column of Table 3 identifies the toxicant of the toxicant-contaminated influent to the reactor. It will be observed that the toxicant employed in a given isolation-enrichment medium is similar to, or closely related to the toxicant, or toxicants found in the influent from which the toxicant is to be reduced, and destroyed.
- Cellite is a registered trademark product of Manville, of Denver, Colorado; and, it it is made of rigid inorganic materials, predominantly silica, produced in several forms: powder, pellet and sphere.
- the Cellite employed in these runs were in the form of spheres, or beads.
- Column 6 gives the concentration of the toxin in the influent to the reactor, the concentration remaining essentially constant in most of the runs, but varying over a 10 ppm to 25 range in the second run.
- the concentration of the toxin in the effluent stream from the reactor, in ppm is given in Column 7 along with the hydraulic retention time in hours.
- the void volume of the reactor is given in milliliters in Column 8, and also the flow rate in milliliters per minute of the stream input to the reactor.
- biocatalytic compositions which contain immobilized whole cells of the microorganisms of this invention, are admirably efficient in time in completing the biotransformation of the toxicants, and provide a final effluent containing only minimal levels of the toxicants.
- the Pseudomonas fluorescens PPr S-4-1 (ATCC SD 904) created to have a genetic predisposition to utililize PCB's as its carbon source effectively lowers the level of PCB's and chlorinated phenols from 50 ppm to between 2.9 ppm and 4.5 ppm with a hydraulic retention time of only 36.2 hours.
- the Pseudomonas fluorescens Chemostat 5 1/2 (ATCC SD 903) reduces the level of chloroethanes from a 10 ppm to 25 ppm to only 0.5 ppm with a hydraulic retention time of only 6.1 hours.
- the Pseudomonas cepacia Sys 2A (ATCC SD 905) reduces the level of phenolic compounds from an influent concentration of 100 ppm to a level of 2.1 ppm with a hydraulic retention time of only 8.9 hours.
- Methylobacter microorganisms i.e., Methylobacter rhodinum SC 971-Red (ATCC 113-X) and Methylobacter species S 971-White (ATCC 138-X)
- Methylobacter rhodinum SC 971-Red ATCC 113-X
- Methylobacter species S 971-White ATCC 138-X
- the solid substrate to which the microorganisms of this invention are attached is porous, and preferably of pore volume of at least 0.2 microns/gram of solids material.
- the pore volume ranges from about 0.2 microns/gram to about 45 microns/gram, more preferably from about 5 microns/gram to about 15 microns/gram of solids material.
- Particle sizes range generally from about 0.5 millimeters (mm) to about 2.0 mm, preferably from about 0.75 mm to about 1.0 mm, in diameter.
- a substrate shaped as a hollow tube, or as a cylinder provides a support suitable for many uses.
- the tube or cylinder can be of virtually any length, with the diameters of such tubular specimens ranging generally from about 30 cm to about 300 cm, preferably from about 75 cm to about 125 cm.
- Biocatalysts formed on such substrates an be employed as fixed beds or moving beds, generally the former.
- the biocatalyst particles are sized in accordance with accepted engineering principles to provide good contact between the effluent and the biocatalytic particles.
- Solid surfaces to which the microorganisms can be affixed are, preferably an aminopolysaccharide surface such as chitan, chitosan, n-carboxy chitosan, cellulose, or a porous inorganic oxide such as alumina, silica, silica-alumina, clay, diatomaceous earth or the like.
- a preferred support is one wherein the chitin, or chitosan is dispersed upon a second solid support, e.g., a porous inorganic oxide.
- the classes of useful porous inorganic oxides is quite large, exemplary of which are, e.g., (1) silica or silica gel, clays, and silicates including tose synthetically prepared and naturally occurring, which may or may not be acid treated, for example, attapulgus clay, china clay, diatomaceous earth, fuller's earth, kaolin, kieselguhr, etc.; (2) ceramics, porcelain, crushed firebrick, bauxite; (3) synthetic and naturally occurring refractory inorganic oxides such as alumina, titanium dioxide, zirconium dioxide, chromium oxide, zinc oxide, magnesia, thoria, boria, silica-alumina, silica-magnesia, chromia-alumina, alumina-boria, silica-zirconia, silica carbide, boron nitride, etc.; (4) crystalline zeolitic aluminosilicates such as naturally occurring or synthetic
- the solid support surface to which the microorganism can be affixed can be used advantageously in the method of this invention in any configuration, shape, or size which exposes a catalytically available amount of the microorganism disposed thereon to the effluent to be treated.
- the choice of configuration, shape, and size of the refractory inorganic oxide depends on the particular circumstances of use of the method of this invention.
- the support surface can be conveniently employed in particulate form, as pills, pellets, granules, rings, spheres, rods, hollow tubes, etc.
- Exemplary of preferred materials of hollow tubular shape are, e.g., Amicon's ultrafiltration membranes cast from a variety of polymer solutions, these consisting of membranes of a very thin (0.1 to 1.5 m), dense "skin" of extremely fine, controlled pore structure which opens to a much thicker (50 to 250 m), open-celled spongy layer of the same polymer, e.g., polysulfone or acrylic copolymers.
- a particulate support form is especially desirable for large volumes of the catalytic composite, and for use in circumstances in which periodic replacement of the biocatalytic composite may be desired.
- Various organic compounds, aldehydes, ketones, acids and the like are suitable for pretreatment and impregnation of a substrate to modify same, and provide a carbon source thereupon or within the substrate to which the microorganisms of this invention can attach, reproduce and grow.
- Chitin, chitosan and cellulose per se contain carbon, and when treated with the organic compound both the organic compound and the chitin, chitosan or cellulose constitute a carbon source for the microorganisms.
- the organic compounds e.g., aldehydes ketones, or acids are applied to the substrate as solutions, generally aqueous solutions, containing from about 0.1 to about 5 percent, preferably from about 0.5 to about 2 percent, of the organic compound, based on the weight of the solution.
- R is H, alkyl, alkenyl, alkynyl, aryl, alkaryl, arylalkyl
- ketones are those characterized by the formula R-R(o)R 1 wherein R and R 1 can be the same or different, and selected from H, alkyl, alkenyl, alkynyl, aryl, alkaryl, arylalkyl groups and the like, e.g., acetone, ethyl propyl ketone, methyl isopropyl ketone, methyl heptyle ketone, methyl vinyl ketone; and ketenes such as dimethylketene, diphenylketene and the like.
- organic acids are e.g., formic acid, acetic acid, butyric acid, capric acid, propiolic acid, cyclobutanecarboxylic acid trimellitic acid, and the like.
- the chitin, chitosan or cellulose substrates are suitably partly digested with the organic compound, in solution, to form a polysaccharide coating by chemical modification of the beta linkages.
- the non-carbon substrates e.g., a diatomaceous earth or porous inorganic oxide, are impregnated and coated by such solutions to form a carbon veneer, coating or carbon sites to which the microorganisms become attached.
- Such treatment with the solution of the carbon compound is generally continued over a period ranging from about 1 to about 48 hours, preferably from about 12 to about 24 hours.
- the microorganism of choise is then contacted with the pretreated substrate by contact therewith of a solution containing the microorganism of choice, to which is added nitrogen, phosphorus, and minerals, at conditions favoring optimum growth of the organism.
- the contact of the solution with the substrate is continued over a period ranging from about 8 hours to about 96 hours, preferably from about 24 hours to about 48 hours.
- a preferred method for adding the microorganisms to the pretreated substrate, or substrate containing the carbon sites is to continously recycle the solution containing the selected microorganism over said pretreated substrate.
- the microorganisms are reproduced, and grow, attaching themselves onto the substrate, becoming matted with the substrate. After formation of the microbial mat the system is generally flushed with water to remove nonattached microorganisms, this exposing and maximizing the active sites on said biocatalytic substrate.
- the biocatalyst composition once formed by affixing the microorganism to the substrate, can be used immediately for detoxification of an effluent containing the toxic substance used for the separation, purification and growth of the respective microorganism, or dried, stored and then reactivated by the addition thereto of a liquid media compatible with the microorganism.
- Various toxicants can be added to soil samples, or sediments, to produce cultures of a microorganism which, when subsequently separated from its natural environment, purified, genetically modified and affixed to a substrate as herein described will form a biocatalyst effective for the detoxification of an effluent containing the toxicant originally used for the production and growth of said microorganism.
- Organochlorides e.g., benzenehexachloride (BHC)
- BHC benzenehexachloride
- Organophenols e.g., phenol
- Organophenols can be used to treat a soil, or sediment from which cultures of phenol-resistant microorganisms can be reproduced, grown and affixed to a substrate, and the substrate containing the phenol-resistant microorganisms then employed as a biocatalyst to detoxify phenol-containing effluents.
- organophenols e.g., 2-chlorophenol, 4-nitrophenol and the like.
- Organophosphates e.g., methyl parathion and azinphosmethyl
- Organophosphates can also be used for the production from a soil, or sediment, of such microorganisms, the microorganisms then attached to a substrate to form a biocatalyst, and the biocatalyst then employed for the detoxification of an effluent containing such toxicant.
Abstract
Description
TABLE 1 __________________________________________________________________________ Pseudomonas Pseudomonas Pseudomonas fluorescens, fluorescens, cepacia, PPr Chemostat Svs S-4-1 51/2 mo 2-A (ATCC SD 904) (ATCC SD 903) (ATCC SD 905) __________________________________________________________________________ Gram reaction - - - shape rod rod rod ONPG (B--galactosidase) - - - arginine dihydrolase + + - lysine decarboxylase - - - ornithine decarboxylase - - - citrate utilization + + + H.sub.2 S production - - - urease - - - tryptophan deaminase - - - indol production - - - Voges-Proskauer - - - gelatinase + + - acid from: glucose - - + mannitol - - - inositol - - - sorbitol - - - rhamnose - - - sucrose - - - melibiose - - - amygdalin - - - arabinose - - - cytochrome oxidase + + + __________________________________________________________________________
TABLE 2 ______________________________________ Physiology & Biochemistry: Methylobacter Methylobacter rhodinum species SC 971-Red SC 971-White (ATCC 113-X).sup.(2) (ATCC 138-X).sup.(3) ______________________________________ motility + - 4 C growth - - 25 C growth + + 30 C growth + + 37 C growth - w 41 C growth - - Pigment diffusible - - Pigment non-diffusible + - Pigment color pink - Pyocyanine produced - - Fluorescein produced - - Melanin pigment - - produced pH 6.0 growth + + 3% NaCl growth - - 6.5% NaCl growth - - MacConkey agar growth - + Skim milk agar growth + + Casein hydrolysis - - Starch hydrolysis - - Gelatinase - - Tween 20 hydrolysis + - Tween 80 hydrolysis - - Indole - - Simmons citrate growth + - Urease + + Nitrate to nitrite - - Nitrite reduction - - Nitrite to nitrogen gas - - Hydrogen sulfide (TSI) - - Lysine decarboxylase - - Arginine (Mollers) - - Ornithine decarboxylase - - Lecithinase - - Catalase + v Oxidase w w Growth on malonate as SCS + - Tyrosine degradation - - Growth on 0.05% cetrimide - - Growth on acetate as SCS - - Testosterone degradation - - PHB accumulation + + Aesculin Hydrolysis - - Arginine Hydrolysis - - Phenylalanine deamination - - dl-hydroxybutyrate - - 3-ketolactose from lactose - - Note: w = slow and weak; ND = no data; v = variable one tube weak one tube -
Sole Carbon Sources in Stanier's Mineral Base: L-arabinose - - cellobiose - - D-fructose + - D-glucose - - lactose - - maltose - - D-mannitol - - L-rhamnose - - D-ribose - + D-sorbitol - - sucrose - - trehalose - - D-xylose - + adonitol - - erythritol - - glycerol + - ethanol + - geraniol - - i-inositol - - sebacic acid + - acetamide - - adipate - - benzoate - - butyrate - - citraconate + - D-gluconate + + M--hydroxybenzoate - - 2-ketogluconate - + DL-lactate + + malate + + pelargonate - - propionate - - quinate - + succinate + + L-+-tartrate - - valerate - - B--alanine - - D-A--alanine - + betaine - - glycine - - L-histidine - + DL-norleucine - - L-proline - w D-tryptophan - - L-valine - - DL-arginine - - benzylamine - - butylamine - - putrescine - - mesoconate - - DL-glycerate + + L-tryptophan - + Methanol + + Fermentation of Carbohydrates: in Hugh & Leifson's O-F Medium Acid from L-arabinose + w Acid from cellobiose - K Acid from ethanol + + Acid from D-fructose + K Acid from D-glucose AO2 - K Acid from D-glucose AnO2 - - Alkaline pH in D-glucose + + Acid from glycerol + K Acid from i-inositol - K Acid from lactose - K Acid from maltose - K Acid from D-mannitol - K Acid from D-mannose - K Acid from L-rhamnose - w Acid from D-ribose + w Acid from sucrose - K Acid from trehalose - K Acid from D-xylose + w control K K ______________________________________ Note 1: w -- weak positive; + = acid; - = no change; K = alkaline Note 2: Cells are nonspore forming gram negative rods. Cells are large and vacular, with one to three polar, subpolar, or lateral flagella. Colonies are entire, smooth and pink. Growth is slow and sparse on Nutrient agar, Trypticase soy agar and BrainHeart Infusion agar. Growth is much better o Pseudomonas F agar, the isolate produces shiny colonies. Note 3: Cells are nonspore forming gram negative rods. Cells are very short fat rods. Motility is absent. Colonies are smooth entire, white and slow growing on Nutrient agar. On BrainHeart Infusion agar colonies become glistening and mucoid.
TABLE 3 __________________________________________________________________________ Influent Effluent Toxicant Environ- Isolation- Toxicant Concentration, Void Flow mental Enrichment Concen- [Hydraulic Retention Volume,/ Rate Microorganism Source Media Toxicants Substrate(s) tration, ppm Time] /mL mL/min __________________________________________________________________________ 1. Pseudomonas Capital Lake, Minimal Salt PCB's and Chitin- 50 4.5-2.9 (±0.5) 117 mL/10 mL/min fluorescens Baton Rouge, Solution and chorinated coated [36.2 hrs] PPr S-4-1 La. (fresh Aroclor ™ .sup.(1) phenols Cellite.sup.(2) (ATCC SD water lake) Mixture 904) 2. Pseudomonas Groundwater Minimal Salt Chlorinated Chitin- 10-25 0.5 (±0.1) 117 mL/10 mL/min fluorescens Sulfur, La. Solution and ethanes coated [6.1 hrs] Chemostat di-or tri di, tri Cellite.sup.(2) 51/2 (ATCC chloroethane SD 903) 3. Pseudomonas Mississippi Minimal Salt phenols, Chitin- 100 2.1 (±0.4) 220 mL/10 mL/min cepacia River Gulf Solution and penta- coated [8.9 hrs] Sys 2A Outlet, Chlorinated chloro- Cellite.sup.(2) (ATCC SD Shell Beach, phenol phenols, 905) La. cresols 4. Methylobacter Activated Minimal Salt triazine Chitin- 50 12 170 mL/1 mL/min rhodinum Charcoal, Solution and herbacides, coated [1240 hrs] SC 971-Red Waste Treat- 50 ppm triazine Cellite.sup.(2) (ATCC 113-X) ment Unit triazine homologues, St. Gabriel, triazine La. ring compounds, RDX compounds 5. Methylobacter Activated Minimal Salt triazine Chitin- 50 12 170 mL/1 mL/min species Charcoal, Solution and herbacides, coated [1240 hrs] SC 971-White Waste Treat- 50 ppm triazine Cellite.sup.(2) (ATCC 138-X) ment Unit triazine homologues, St. Gabriel, triazine La. ring compounds, RDX compounds (1,3,5- trinitro- 1,3,5- triazine) __________________________________________________________________________ Note.sup.1 Aroclor ™ is a commercial mixture of Aroclor 1232, Aroclor 1248, and Aroclor 1254 obtained from Foxboro Analabs. These isomer/cogeno mixtures represent an adequate sampling of the possible 209 PCB combinations, including specifically 4,4' dichlorobiphenyl (4,4' DCB), 2,5,4' trichlorobiphenyl (2,5,4' TCB) and 2,4,5,2',5' pentachlorobiphenyl (2,4,5,2',5' PCB). Note.sup.2 Cellite, a registered trademark of Manville. A rigid inorgani catalyst carrier, principally of silicous matter. Cellite employed was Cellite R630 spheres.
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US06/918,840 US4859594A (en) | 1986-10-14 | 1986-10-14 | Microorganisms for biodegrading toxic chemicals |
US07/003,019 US4775650A (en) | 1986-10-14 | 1987-01-13 | Decontamination of contaminated streams |
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